Expression systems for transcription of functional nucliec acid

ABSTRACT

A ribozyme comprising the following base sequence (I) or (II):  
                     base sequence (I)(SEQ ID No. 1):         5′-ACCGUUGGUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCUAACACGC           GAAAGGUCCCCGGUUCGAAACCGGGCACUACAAACACAACACUGAUGAGG           ACCGAAAGGUCCGAAACGGGCACGUCGGAAACGGUUUU[[U]]-3′           base sequence (II)(SEQ ID No. 2):     5′-ACCGUUGGUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCUAACACGC           GAAAGGUCCCCGGUUCGAAACCGGGCACUACAAACCAACACACAACACUG           AUGAGGACCGAAAGGUCCGAAACGGGCACGUCGGAAACGGUUU           U[[U]]-3′.

TECHNICAL FIELD

[0001] The present invention relates to ribozymes and their expressionsystems.

BACKGROUND ART

[0002] A hammerhead ribozyme is one of the smallest catalytic RNAMolecules (Kruger et al., 1982; Guerrier-Takada et al., 1983). Becauseof its small size and potential as an antiviral agent, numerousmechanistic studies (Dahm and Uhlenbech, 1991, Dahm et al., 1993;Eckstein and Lilley, 1996; Pontius et al., 1997; Lott et al., 1998; Zhouet al., 1996, 1997; Zhou and Taira, 1998) and studies directed towardsapplication in vivo have been performed (Erickson and Izant, 1992;Murray, 1992; Rossi, 1995; Eckstein and Lilley, 1996; Prislei et al.,1997; Turner, 1997; Scanlon, 1997). Many successful experiments, aimedat the use of ribozymes for suppression of gene expression in differentorganisms, have been reported (Sarver et al., 1990; Dropulic et al.,1992; Ojwang et al, 1992; Yu et al, 1993; Zhao and Pick, 1993; Inokuchiet al, 1994; Yamada et al, 1994; Ferbeyre et al, 1996; Fujita et al,1997; Kawasaki et al, 1998). However, the efficacy of ribozymes in vitrois not necessarily correlated with functional activity in vivo. Some ofthe reasons for this ineffectiveness in vivo are as follows. i) Cellularproteins may inhibit the binding of the ribozyme to its target RNA ormay disrupt the active conformation of the ribozyme. ii) Theintracellular concentration of metal ions essential forribozyme-mediated cleavage might not be sufficient for functionalactivity. iii) Ribozymes are easily attacked by RNases. However, we arenow starting to understand the parameters that determine ribozymeactivity in vivo (Bertrand and Rossi, 1996; Bertrand et al., 1997;Gebhard et al., 1997). Studies in vivo have suggested that the followingfactors are important for the effective ribozyme-mediated inactivationof genes: a high level of ribozyme expression (Yu et al., 1993); theintracellular stability of the ribozyme (Rossi and Sarver, 1990;Eckstein and Lilley, 1996); co-localization of the ribozyme and itstarget RNA in the same cellular compartment (Sullenger and Cech, 1993;Bertrand et al., 1997); and the cleavage activity of the transcribedribozyme (Thompson et al., 1995). Recently, it was shown that thesevarious features depend on the expression system that is used (Bertrandet al., 1997).

[0003] The RNA polymerase II (pol II) system, which is employed fortranscription of mRNAs, and the polymerase III (pol III) system,employed for transcription of small RNAs, such as tRNA and snRNA, havebeen used as ribozyme expression systems (Turner, 1997). Transcriptsdriven by the pol II promoter have extra sequences at the 3′ and 5′ ends(for example, an untranslated region, a cap structure, and a polyAtail), in addition to the coding region. These extra sequences areessential for stability in vivo and functional recognition as mRNA. Atranscript containing a ribozyme sequence driven by the pol II promoterincludes all those sequences, unless such sequences are trimmed aftertranscription (Taira et al., 1991; Ohkawa et al., 1993). As a result, insome case, the site by which the ribozyme recognizes its target may bemasked, for example, by a part of the coding sequence. By contrast, thepol III system is suitable for expression of short RNAs and only veryshort extra sequences are generated. In addition, expression is at leastone order of magnitude higher than that obtained with the pol II system(Cotten and Birnstiel, 1989). Thus, it was suggested that the pol IIIsystem might be very useful for expression of ribozymes (Yu et al.,1993; Perriman et al., 1995). However, in many cases, the expectedeffects of ribozymes could not be achieved in spite of the apparentlydesirable features of the pol III system (Ilves et al. 1996; Bertrand etal., 1997)

DISCLOSURE OF THE INVENTION

[0004] In order to investigate the parameters that determine ribozymeactivity in vivo, we designed three types of ribozyme with an identicalribozyme sequence, driven by tRNA^(Val) promoter which is a pol IIIpromoter, and demonstrated that the entire structure of the transcript(ribozyme to which the sequence of tRNA^(Val) is added (hereinaftertermed “tRNA^(val)-ribozyme”)) determined not only cleavage activity butalso the intracellular half-life of the ribozyme. All the chimerictRNA^(Val)-ribozymes that were transcribed in the cell nucleus wereexported to the cytoplasm. Thus, the ribozymes and their target werepresent within the same cellular compartment. Under these conditions, wefound that the intracellular half-life and-the steady-state level ofeach. tRNA^(val)-ribozyme were the major determinants of functionalactivity in vivo. Moreover, we demonstrated that cells that expressed aspecifically designed ribozyme with the longest half-life in vivo werealmost completely resistant to a challenge by HIV-1. Further, byestablishing a small bulge structure (“bulge” refers to, in the casewhere RNA adopts a hairpin structure, a portion where there is aprotruding single-stranded structure of unmatched base pairs) at theamino-acyl stem portion of the tRNA^(Val) structure, avoidance ofrecognition from the mature enzyme can be achieved and as a result, anyRNA sequence comprising a ribozyme sequence connected to the 3′ end canbe made to exist intracellularly in a form where it is connected totRNA^(Val). Any RNA comprising a ribozyme sequence connected to the 3′end of the tRNA^(Val) structure of the present invention, due to theproperties of the tRNA structure, is transported stably and efficientlyto the cytoplasm. This is of particular importance for the intracellularfunction of the ribozyme.

[0005] A summary of the present invention is presented as follows:

[0006] 1. A ribozyme comprising a nucleotide sequence having thefollowing base sequence (I) or (II): base sequence (I):5′-ACCGUUGGUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCUAACACGCGAAAGGUCCCCGGUUCGAAACCGGGCACUACAAACACAACACUGAUGAGGACCGAAAGGUCCGAAACGGGCACGUCGGAAACGGUUUUU-3′ base sequence (II):5′-ACCGUUGGUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCUAACACGCGAAAGGUCCCCGGUUCGAAACCGGGCACUACAAACCAACACACAACACUGAUGAGGACCGAAAGGUCCGAAACGGGCACGUCGGAAACGGUUUUU-3′

[0007] 2. An expression vector comprising DNA encoding the ribozymeaccording to 1 above.

[0008] 3. A method of producing the ribozyme according to 1 abovecomprising transcribing to RNA with expression vector DNA as a template,wherein said expression vector DNA comprises DNA encoding the ribozymeaccording to 1 above.

[0009] 4. A pharmaceutical composition comprising the ribozyme accordingto 1 above or the expression vector according to 2 above, as aneffective ingredient.

[0010] 5. The pharmaceutical composition according to 4 above for theprevention and/or treatment of acquired immune deficiency syndrome.

[0011] 6. A method of specifically cleaving a target RNA using theribozyme according to 1 above.

[0012] 7. The method of 6 above wherein the target RNA is HIV-1 RNA.

[0013] 8. An RNA variant (mature tRNA^(Val)) adopting the followingsecondary structure (I), wherein said RNA variant comprises a bulgestructure introduced in the region in which hydrogen bonds form betweennucleotides 8 to 14 and nucleotides 73 to 79.

[0014] 9. The RNA variant of 8 above wherein a bulge structure isintroduced by substituting all or part of the sequence of the regioncorresponding to nucleotides 73 to 79 within a nucleotide sequence of anRNA adopting secondary structure (I).

[0015] 10. The RNA variant according to 8 above consisting of thesequence of a region corresponding to nucleotides 1-80 within anucleotide sequence represented by SEQ ID NO: 1.

[0016] 11. The RNA variant according to 8 above consisting of thesequence of a region corresponding to nucleotides 1-86 within anucleotide sequence represented by SEQ ID NO: 2.

[0017] 12. An RNA comprising the RNA variant of 8 above and a selectedRNA chain linked thereto.

[0018] 13. The RNA according to 12 above wherein selected RNA chain is aribozyme or an antisense RNA.

[0019] 14. The RNA according to 12 above wherein a bulge structure isformed with any nucleotide of an RNA chain linked to the 3′ terminus andany nucleotide of the region of nucleotides 8 to 14 within thenucleotide sequence of an RNA adopting secondary structure (I).

[0020] 15. An expression vector comprising DNA encoding the RNA of 12above.

[0021] Having consideration for the transcription amount, stability andpost-transcription activity of ribozymes, we selected human tRNA^(Val)promoter which is involved in a polymerase III system, as an expressionsystem therefor, and examined whether there was any difference inribozyme effect in vivo due to the way in which the ribozyme was linkedto this promoter. In other words, we focussed on intracellular stabilitywhich is an important factor in obtaining significant ribozyme effect invivo, and post-transcription activity, and set out to clarify therelationship between the high-order structure of ribozymes and thesefactors.

[0022] First, we designed a hammerhead ribozyme targeting a relativelyconserved sequence of HIV-1, and constructed four expression systems byattaching this gene to downstream of the tRNA^(Val) promoter via varioussequences. As a vector for the construction of these expression systemswe used pUC19 (Takara), however, other vectors such as PGREEN LANTERN(Life Technologies Oriental, Inc.) and pHaMDR (HUMAN GENE THERAPY6:905-915 (July 1995)) may also be used. Also, oligonucleotide sequencesnecessary for the construction of these expression systems can bechemically synthesized with a DNA/RNA synthesizer (Model 394; AppliedBiosystems, Division of Perkin Elmer Co. (ABI), Foster City, Calif.).

[0023] From predictions made using Zuker's method, it was thought thatdifferences in the linker sequence used to connect the tRNA^(Val)promoter and hammerhead ribozyme would exert great influence on thesecondary structure of the recognition site of the ribozyme (See FIG.1). According to this prediction map, it was clear that whereas theoverall secondary structure of the ribozyme was almost the same, thedegree of freedom at the substrate-binding site differed greatly. It isclear that whereas both substrate binding sites form a stem structurewithin the molecule in Rz1, one binding site in Rz2, and both bindingsites in Rz3 protrude to the outside. In the case of Rz3, the protrudingsubstrate binding site may be masked by protein. However, since aribozyme is an RNA enzyme and both binding ability and disassociationability with a substrate are important factors in its activity, Rz3 wasexpected to be the best in terms of cleavage ability. We performed areaction using intracellularly transcribed. ribozymes, in an in vitrosystem under the following conditions: 40 mM Tris-Cl (pH8.0), 8 mMMgCl₂, 5 mM DTT, 2 mM Spermidine, 2 U/μl RNase inhibitor, 30 μg totalRNA. At this time, the ribozyme content in total RNA was made constant.The results showed that ribozyme activity toward short substrates thatwere transcribed in vitro and radioactively labeled depended on thedegree of freedom at the recognition site (See FIG. 2). Further,stability of the ribozymes was examined. With the expression amount of acontrol gene made constant, we conducted a comparative study of eachribozyme amount. The difference in ribozyme structure also affectedstability. The reason why structures having such little overalldifference exert this influence is not clear, however, a difference ofapproximately 25 times was exhibited as between the most stable and theleast (FIG. 3B).

[0024] We next examined the relationship between ribozyme activity invitro and in vivo effect, which as discussed above is as yet unclear.First, using a luciferase gene as a reporter gene, a system forevaluating ribozyme effect was constructed wherein ribozymes are allowedto act on a fusion gene of this reporter gene and the sequence pNL4-3(an HIV-1 clone), and luciferase activity in the cell extract ismeasured (See FIG. 3A). A comparison of each of the ribozymes showedthat the one with the highest intracellular stability exhibited highestactivity suggesting the importance of stability (See FIG. 4).

[0025] The above discussion relates to an evaluation of ribozyme effecton an artificial fusion gene of a luciferase gene and a HIV-1 sequence,in cultured cells. Therefore, it may be difficult to judge that theseresults are the equivalent of results that could be obtained in anorganism. Thus, we performed an evaluation of ribozyme effect againstactual HIV-1 (See FIG. 5). A ribozyme expression system transformant wasinfected with HIV-1, and virus growth was measured by measuring theamount of p24 (core protein of the virus) produced in serum. Resultsindicating a similar trend to our evaluation in cultured cells wereobtained. Further, it was clear that the ribozyme with the highest invitro stability exhibited very high inhibitory effect with production ofp24 inhibited by 99% (See FIG. 6C) In contrast, the ribozyme expressionsystem having the highest cleavage activity in vitro was mostly unableto inhibit growth of the virus.

[0026] In this manner it became clear that the evaluation of ribozymeeffect with the virus indicated the same trend as the evaluation withthe artificial substrate in cultured cells. Thus, the results of theonce-over evaluation we performed are thought to be a rough guide to theeffect of the ribozymes in vivo. Further, from the results of theseexperiments both in vitro and those dealing with a virus, it is clearthat to obtain significant intracellular effect of the ribozyme, whileactivity is important, intracellular stability is of greater importance.The fact shown in the case described herein, that even ribozymes whosesequences have little differences lead to great differences in effect invivo depending on the linkage to the expression system, must be fullyconsidered. The above result also suggests that it is important todesign ribozymes with high stability while considering the influence ofhigher structures comprising added sequences for expression on theirstability.

[0027] tRNA is first recognized intracellularly with its promotersequence consisting of sequences known as A box and B box within itsstructural sequence, and transcribed with the extra sequences connectedto the 5′ and 3′ ends. Next, with to the action of a plurality of matureenzymes which exist within cells, extra sequence are eliminated to formmature tRNA. In the action of these enzymes, the structure of theportion known as the amino-acyl stem becomes an important determinant ofstructure recognition. We found that by establishing a small bulgestructure at the portion, it was possible to avoid the action of themature enzyme. For example, in FIG. 1, each of Rz1, Rz2 and Rz3 has asmall bulge structure at the amino-acyl stem, and as a result, theribozyme sequence of the transcribed RNA is not eliminated (FIGS. 3B, 7Aand determined base sequence). Such a property is not due to the 3′ endsequence being a ribozyme but is due to the bulge structure at theamino-acyl stem portion, consequently, any RNA sequence comprisingantisense may be used.

[0028] Typically tRNA undergoes a series of processings beforetransportation from the nucleus including the removal of extra sequencesat the 5′ and 3′ ends by a mature enzyme, removal of any introns bysplicing mechanism, modification of specific bases, and addition of asequence consisting of 5′-CCA-3′ to the 3′ terminus and, in some casesaddition of amino acids suitable for said tRNA (amino-acylation).

[0029] However, the tRNA of the present invention is activelytransported out of the nucleus without being subject to, from among theabove series of modifications, at least, removal of extra sequences atthe 5′ and 3′ ends, addition of a CCA sequence to the 3′ end, andsubsequent amino acylation. (FIG. 7A). This is likely becauseestablishing a bulge in the amino acyl stem portion results in avoidanceof action by mature enzymes and following addition of CCA sequencefollowed by amino acylation, and the entire structure resembles that ofthe original tRNA. This inference was also supported by the fact thatthe one with degenerated tRNA structure (Rz4), according to structurepredictions using computers, was not transported to the cytoplasm. (FIG.7C). This property of being transported from the nucleus to thecytoplasm does not depend on the ribozyme sequence at the 3′ end, andthus it is thought that any RNA sequence such as antisense RNA may beused similarly.

[0030] In recent years, it has come to be understood that whereantisense RNA and ribozyme RNA are expressed intracellularly, in orderto elicit their function, it is important that their distribution in acell is within the cytoplasm. Since RNAs of the present invention formstable tRNA-like structures by themselves, they have the function ofdefinitely transporting to the cytoplasm without exerting greatinfluence on the higher-order structure of the RNA linked to the 3′ end(which is an extremely important property when the RNA linked to the 3′end is a functional RNA such as a ribozyme or antisense RNA). Further,when the RNA is made into DNA, it functions as a promoter irrespectiveof cell type and it has a broad range of hosts (originally of humanderivation, but should be able to express in at least all mammals.) Inshort, the present invention is most suitable for an antisense RNA andribozyme RNA expression systems, and can become an important tool forexperiments using cultured cells and for gene therapy in the field ofmedicine. Further, through the technique of molecular evolutionengineering, RNA molecules with functions not found in nature, arerecently being artificially created. If these molecules exhibit theirfunctions intracellularly, particularly in the cytoplasm, then thepresent invention will be able to be used as an expression system forthese RNA molecules.

[0031] Using the tRNA^(val)-ribozyme of the present invention it ispossible to specifically cleave a target RNA, particularly an HIV-1 RNA.

[0032] The tRNA^(val)-ribozyme of the present invention can be used as amedicine especially for the prevention and/or treatment of acquiredimmune deficiency syndrome. For example, the transcription of HIV can beinhibited by encapsulating the tRNA^(Val)-ribozyme of the presentinvention in a liposome, administering this to an organism and allowingincorporation into cells comprising HIV. Further, transcription of HIVcan be inhibited by incorporating DNA encoding the tRNA^(Val)-ribozymeof the present invention in a vector such as virus, introducing thevector into cell comprising HIV to allow intracellular expression of thevector thereby effecting production of the tRNA^(Val)-ribozyme of thepresent invention. Administration of the tRNA^(Val)-ribozyme of thepresent invention will depend on severity of the conditions of thepatient and responsiveness of the organism, and may be conducted inappropriate amount, form of administration and frequency, and over aperiod until the efficacy of prevention and/or treatment can berecognized, or until alleviation of the patient's condition is achieved.

[0033] The present specification incorporates in its entirety thecontent of the specification and drawings of Japanese Patent ApplicationNo. 10-244755, said application forming the basis of the priority claimof this application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 shows the secondary structures of tRNA^(Val)-ribozymes thatwere predicted by computer folding. The sequence of hammerhead ribozyme(bold capital letters) was ligated with that of tRNA^(Val) sequence(capital letters) by means of various linker sequences. The sequencesthat correspond to the internal promoter of seven-base-deletedtRNA^(Val), namely the A and B boxes, are indicated by shaded boxes.Diagrams A to D show the secondary structures of tRNA^(Val)-ribozyme 1(Rz1), 2 (Rz2), 3 (Rz3) and 4 (Rz4), respectively. The recognition armsof ribozymes are indicated by underlining. Diagram E shows the secondarystructure of the transcript of human placental tRNA^(Val). The tRNA isprocessed at three sites (arrowheads) to yield in the mature tRNA^(Val)(capital letters).

[0035]FIG. 2 indicates the cleavages mediated by tRNA^(Val)-ribozyme invitro. Panel A is a schematic representation of the substrate RNA (thesubstrate RNA corresponds to nucleotides 500-711 of pNL432, namely theU5 region of HIV-1 RNA). The substrate RNA was cleaved into twofragments by the tRNA^(Val)-ribozyme (5′ fragment, 70-mer; 3′ fragment156-mer). Panel B is an autoradiogram showing the results of cleavagereactions. Lanes; M, markers; vector, tRNA^(Val) vector alone without aribozyme; Rz1-ribozyme 1; Rz2-ribozyme 2; and Rz3-ribozyme 3.

[0036]FIG. 3 indicates the stability of tRNA^(Val)-ribozyme in vivo.Panel A is a schematic representation of pUC-Rr that allowednormalization of the efficiency of transfection by the use of areference gene. The reference gene was expressed downstream of theribozyme-expression cassette. The sequences of the promoter andterminator were the same, respectively, in the two expression cassettes.Panel B shows steady-state levels of expression of tRNA^(Val)-ribozyme.This figure shows Northern blotting analysis with the probe specific forthe ribozyme (upper) and for the reference gene (bottom). FIG. Cindicates the half-lives of tRNA^(Val)-ribozymes in stablyribozyme-transduced cells. The circles indicate relative amounts oftRNA^(Val)-ribozyme 1 (Rz1). Squares and diamonds indicate relativeamounts of ribozyme 2 (Rz2) and 3 (Rz3), respectively. Bars show S.E. ofresults from 3 assays.

[0037]FIG. 4 shows the inhibition of production of the U5 LTR-luciferasefusion gene in HeLa cells. Panel A. Transient expression in HeLa cells.Both the target-expressing plasmid and pUCdt-Rz encoding a ribozyme wereused to co-transfect HeLa cells. Panel B. Transient expression in stablyribozyme-transduced cells. Two independent clones were selected for eachconstruct with similar levels of transcription of the inserted gene(tRNA^(Val) or tRNA^(Val)-ribozyme). Only the target-expressing plasmidwas used to transfect ribozyme-producing HeLa cells. Bars show S.E. ofresults from 5 assays.

[0038]FIG. 5 is a schematic representation of the HIV vector. Theexpression cassette for each tRNA^(Val)-ribozyme was inserted into theSalI site immediately upstream of TK-neo^(r) in HIV-I-derived vector (A)to yield a retroviral vector, HIVRibo.N, that encoded atRNA^(Val)-ribozyme(B). Ψ indicates a packaging signal.

[0039]FIG. 6 shows quantitation of the expression of tRNA^(Val)-ribozymein stably ribozyme-transduced H9 cells (CD4+ T cells) and inhibition ofproduction of p24 in the transduced cells. Panel A. Quantitation ofresults, shown in B, of Southern blotting analysis of theRT-PCR-amplified ribozyme from two independent clones ofribozyme-transduced H9 cells. Products of PCR after 13, 15, and 17cycles were analyzed by Southern blotting using a ³²P-labeledoligonucleotide probe. Squares and circles indicate the results withtransduced cells of the ribozyme 2 (Rz2) and the ribozyme 3 (Rz3)respectively. Panel B indicates the results of Southern blotting. PanelC. Cells were cultured for 11 days after infection with HIV-1 NL432.Small aliquots of supernatant were prepared from each culture on days 3,7, and 11. Levels of p24 antigen were determined by HIV-1antigen-capture ELISA. The triangles indicate the result oftRNA^(Val)-ribozyme 1 (Rz1) Squares and circles indicate the resultswith the ribozyme 2 (Rz2) and ribozyme 3 (Rz3), respectively. Trianglesindicate the results with the control.

[0040]FIG. 7 shows intracellular localization of thetRNA^(Val)-ribozyme. Northern blotting analysis was performed using RNAfrom each intracellular fraction. Nucleic and cytoplasmic RNAs wereprepared separately from cells that had been stably transduced with thegene for a ribozyme (the tRNA^(Val)-ribozyme-producing HeLa cells usedin the experiments for which results are given in FIG. 4B). A and C showthe results obtained with a ³²P-labeled probe specific for thetRNA^(Val)-ribozyme. B and D show controls: Contamination of thecytoplasmic fractions was examined with a probe specific for thetranscript of the natural U6 gene.

BEST MODE FOR CARRYING OUT THE INVENTION

[0041] Below, the present invention will be explained in furtherdetailed by means of examples. However, this is not intended to limitthe scope of the present invention to these examples.

EXAMPLES

[0042] Materials and Methods

[0043] Construction of Plasmids

[0044] The plasmids (pUCdt-Rz series) that expressed eachtRNA^(Val)-ribozyme were constructed as follows. Both sense andantisense oligonucleotide linkers encoding the sequence of the promoterregion, derived from the human gene for placental tRNA^(Val)(pHtV1;Arnold et al., 1986), were annealed and ligated into the EcoRI/SalI siteof pUC19. The sequences of the oligonucleotide linkers were as follows:sense, 5′-aat tca gga cta gtc ttt tag gtc aaa aag aag aag ctt tgt aaccgt tgg ttt ccg tag tgt agt ggt tat cac gtt cgc cta aca cgc gaa agg tccccg gtt cga ag-3′ (SEQ ID NO: 6); antisense, 5′-tcg act tcg aac cgg ggacct ttc gcg tgt tag gcg aac gtg ata acc act aca cta cgg aaa cca acg gttaca aag ctt ctt ctt ctt ttt gac cta aaa gac tag tcc tg-3′ (SEQ ID NO:7). Next, both sense and antisense oligonucleotide linkers that encodedthe terminator sequence were also annealed and ligated into theNspV/SalI site of pUC19 that contained the sequence of the promoterregion. The sequences of oligonucleotide linkers were as follows: sense,5′-cga aac cgg gca ccc ggg gaa tat aac ctc gag cgc ttt ttt tct atc gcgtc-3′ (SEQ ID NO: 8); antisense, 5′-tcg acg cga tag aaa aaa agc gct cgaggt tat att ccc cgg gtg ccc ggt ttc-3′ (SEQ ID NO: 9). The resultantplasmid which contained the A and B boxes of tRNA^(Val) and aterminator, was designated pUCdt.

[0045] DNA fragments encoding the sequence of each ribozyme and thetRNA^(Val) portion were amplified by PCR using pUCdt as a template withan upper primer (5′-cgc cag ggt ttc cca gtc acg ac-3′) (SEQ ID NO: 10)and a lower primer that included the sequences of both the ribozyme andthe terminator (Rz1, 5′-ctg cag gtc gac gcg ata gaa aaa aag cgc tcg aggtgc ccg ttt cgt cct cac gga ctc atc agt gtt gtg tgg gtg ccc ggt ttc gaaccg gga cct tt-3′ (SEQ ID NO: 11); Rz2, 5′-ctg cag gtc gac gcg ata gaaaaa aac cgt ttc cga cgt gcc cgt ttc ggt cct ttc ggt cct cat cag tgt tgtgtt tgt agt gcc cgg ttt cga acc ggg gac ctt t-3′ (SEQ ID NO: 12); Rz3,5′-ctg cag gtc gac gcg ata gaa aaa aac cgt ttc cga cgt gcc cgt ttc ggtcct cat cag tgt tgt gtg ttg gtt tgt agt gcc cgg ttt cga acc ggg gac cttt-3′ (SEQ ID NO: 13)). After digestion of products of PCR with the EcoRIand SalI, each fragment was ligated into the EcoRI/SalI site of pUC19 toyield pUCdt-Rz. The sequences of pUCdt and pUCdt-Rz series wereconfirmed by direct nucleotide sequencing. The members of pUC-Rr series,which contained a reference gene-expression cassette in addition to thegene for the tRNA^(Val)-ribozyme (see FIG. 3A), were constructed byinserting the PvuII fragment of pUCdt into the HincII site of eachpUCdt-Rz. The direction of the inserted fragment was confirmed bydigestion with restriction enzymes. The pHyg dt-Rz series, which wasused for generation of the ribozyme-transduced HeLa cells, wasconstructed by inserting each PvuII-SalI fragment of the pUCdt-Rz seriesinto the EcoRV/SalI site of pHyg (Yates et al., 1984). Alloligonucleotide linkers and primers for PCR were synthesized by aDNA/RNA synthesizer (model 392; PE Applied Biosystems, Foster City,Calif.).

[0046] Recombinant HIV vector plasmids were constructed as follows. A2.0-kbp BamHI fragment that encoded the bacterial neo^(r) gene cassettefrom pMC1 neo (Thomas and Capecchi, 1987) was inserted into the SalIsite of an HIV-1-derived vector (FIG. 5A; Shimada et al., 1991). Then,the tRNA^(Val)-ribozyme expression cassette was cloned into the SalIsite, immediately upstream of TK-neo^(r), as shown in FIG. 5B.

[0047] Culture and Transfection of Cells

[0048] HeLa and Cos cells were cultured in Dulbecco's modified Eagle'smedium (DMEM; Gibco BRL, Gaithersburg, Md. supplemented with 10% (v/v)fetal bovine serum (FBS; Gibco BRL) and 45 μg/ml gentamycin (Gibco BRL).To select ribozyme-transduced cells, hygromycin B was used at a finalconcentration of 300 μg/ml. H9 cells were cultured in RPMI (Gibco BRL)supplemented with 10% fetal calf serum (FCS; Gibco BRL).

[0049] Cells were transfected using the Lipofectin reagent (Gibco BRL)according to the manufacturer's protocol. A recombinant HIV vectorplasmid (HIVRib.N; shown in FIG. 5B) was used to transfect H9 cells bythe CaPO, co-precipitation.

[0050] Preparation of RNA

[0051] Total RNA was extracted by the guanidiniumthiocyanate-phenol-chloroform-method. Cytoplasmic RNA and nuclear RNAwere separated as described previously (Huang and Carmichael, 1996).

[0052] Measurement of the Steady-State Levels and Half-Lives ofRibozymes

[0053] The steady-state level of each ribozyme was measured as follows.HeLa cells (1×10⁶ cells/10-cm plate) were transfected with each pUC-Rr.Two days after transfection, total RNA was isolated from these cells.The amount of the reference RNA, located downstream oftRNA^(Val)-ribozyme in the isolated total RNA, was quantified first byNorthern blotting analysis with a probe specific for the reference RNA(5′-aaa tcg cta taa aaa gcg ctc gag gtt atg ctc ccc ggg t-3′) (SEQ IDNO: 14). The amount of the reference DNA in each sample was maintainedat a constant value and the level of total RNA in each sample was alsokept constant by addition of RNA isolated from untransfected HeLa cellsas necessary. Finally, hybridization was repeated with a probe specificfor the ribozyme (5′-ctc atc tgt gtt gtg t-3′) (SEQ ID NO: 15) or theprobe specific for the reference RNA (FIG. 3B).

[0054] The half-life of each ribozyme was determined by Northernblotting analysis after treatment of cells with actinomycin D asdescribed previously (Huang and Gerlach, 1996). In brief, cells wereexposed to actinomycin D at a final concentration of 5 μg/ml for 0, 60,120 or 180 min and, at each time point, total RNA was isolated (FIG.3C). The amount of ribozyme in each preparation of isolated RNA wasdetermined by Northern blotting.

[0055] Cleavage Assay

[0056] Total RNA was isolated from HeLa cells transfected with eachpUCdt-Rz or pUCdt. The amount of ribozyme in each preparation ofisolated RNA was determined by Northern blotting with the probe that wasspecific for the ribozyme. Then the concentration of each ribozyme wasadjusted to the same value by addition of RNA isolated fromuntransfected HeLa cells. The substrate RNA that encoded the U5 LTRregion of HIV-1 (FIG. 2A) was prepared by T7 transcription andradiolabeled with ³²P. Cleavage reactions were allowed to proceed in a50-μl reaction mixture [40 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, 5 mMdithiothreitol (DTT), 2 mM Spermidine, 40 U of placental RNaseinhibitor, 30 μg of total RNA, 5 kcpm of radiolabeled substrate RNA] at37° C. for 12 h. Products were identified after electrophoresis on 6%polyacrylamide gel/7M urea gel (FIG. 2B).

[0057] Luciferase Assay

[0058] Luciferase activity was measured with the Dual-Luciferase™Reporter Assay System (Promega, Madison, Wis.) according to themanufacturer's protocol. HeLa cells transfected with pUCdt-Rz and thetarget-expressing plasmid (FIG. 4A) or ribozyme-producing HeLa cellsthat had been transduced with the target-expressing plasmid (FIG. 4B)were lysed in 150 μl of 1× Passive lysis buffer for 15 min and scrapedoff the plate. The cell debris was removed by centrifugation. Afteraddition of 20 μl of the centrofuged lysate to 100 μl of LuciferaseAssay Reagent II, the luminescent signal was immediately quantitatedwith a luminometer (Lumant LB 9501; Berthold, Bad Wildbad, Germany).Furthermore, for normalization of the activity of firefly luciferase, wemeasured the luminescent signal generated by Renilla luciferase byadding 100 μl of Stop & Glo™ Reagent to the sample tube immediatelyafter quantitation of the reaction catalyzed by firefly luciferase. Therecorded value of firefly luciferase activity was normalized byreference to the activity of Renilla luciferase (FIG. 4).

[0059] Each normalized value of firefly luciferase activity was furthernormalized by reference to the concentration of protein in the lysate.The protein was quantitated with a Protein Assay Kit (Bio-Rad,California, USA) which was based is Bradford's method.

[0060] HeLa Cells stably Transduced with a Ribozyme

[0061] Ribozyme-transduced cells were obtained by transfecting HeLacells with pHyg dt or a member of the pHyg dt-Rz series and selection inDMEM that contained 300 μg/ml hygromycin B (Wako Chemicals, Osaka,Japan). Twelve h after transfection, the medium was replaced by growthmedium and the cells were cultured for another 48 h. The cells weresubcultured at a dilution of 1:5 in DMEM that contained 300 g/mlhygromycin B (selection medium). The medium was replaced by fresh mediumevery three days. Cells resistant to hygromycin B were expanded in DMEMthat contained 250 μg/ml hygromycin B.

[0062] Production of Virus and Transduction of the Ribozyme by an HIVVector

[0063] A supernatant containing recombinant virus was produced asdescribed previously (Shimada, 1991). Cos cells (2×10⁶ cells/10-cmdish)were cultured and transfected with 10 μg of the packaging vectorplasmid and 10 μg of the recombinant HIV vector plasmid (HIVRib.N; shownin FIG. 5). The supernatant, which contained recombinant virus, wascollected after 48 h and filtered through a 0.22 μm pore filter. Then2×10⁶ H9 cells were incubated with 5 ml of the filtered supernatant thatcontained 6 μg/ml Polybrene™ (Abbott Laboratories). After 24 h, themedium was replaced with RPMI supplemented with 10% FCS and 1 mg/mlG418. These cells were cultured for a further 48 h and thenG418-resistant clones were isolated. The transduction of the ribozymegene was confirmed by RT-PCR analysis.

[0064] Quantitation of tRNA^(Val)-Ribozyme Produced in H9 Cells

[0065] Quantitative RT-PCR was carried out as follows (Ozawa et al.,1990; Hamblet and Castora, 1995). Total RNA was extracted from H9 cellsthat had been stably transduced with a ribozyme. cDNA was synthesized ina 20-μl reaction mixture [1 μg of total RNA, 20 mM Tris-HCl (pH 8.3), 50mM KCl, 5 mM MgCl₂, 1 mM dNTP, 1 pmol of primer(for β-actin, 5′-gtg gccatc tct tgc tcg aa-3′ (SEQ ID NO: 16); for ribozyme: 5′-gac ctt tcg gtcctc atc-3′ (SEQ ID NO: 17)) and 0.25 U/ml Moloney murine leukemia virusRtase (Takara Shuzo, Kyoto, Japan)) at 42° C. for 30 min.

[0066] cDNA for β-actin was amplified by PCR with two oligonucleotideprimers (upper, 5′-gac tac ctc atg aag atc ct-3′ (SEQ ID NO: 18); lower:5′-gtg gcc atc tct tgc tcg aa-3-′ (SEQ ID NO: 19)) with 13, 15 or 17cycles of 94° C. for 1 min, 60° C. for 1 min and 72° C. for 2 min.Ribozyme cDNA was amplified by PCR with two oligonucleotide primers(upper, 5′-gtt atc acg ttc gcc taa-3′ (SEQ ID NO: 20); lower: 5′-gac ctttcg gtc ctc atc-3′ (SEQ ID NO: 21)) with 13, 15 or 17 cycles of 94° C.for 1 min, 55° C. for 1 min, and 72° C. for 2 min.

[0067] Products of PCR after 13, 15 and 17 cycles were analyzed bySouthern blotting with a radiolabeled probe specific for the ribozyme(5′-acg cga aag gtc ccc ggt-3′ (SEQ ID NO: 22)) or for β-actin (5′-gcggga aaa tcg tgc gtg a-3′ (SEQ ID NO: 23)). The radioactivity of eachband (FIGS. 6A and 6B) was measured with BAS2000 system (Fuji Film,Tokyo, Japan).

[0068] HIV-1 Challenge Assay

[0069] H9 cells transduced with the ribozyme by the HIV vector(HIVRib.N) and mock-tranduced control cells were incubated with NL432 ata m.o.i. (multiplicity of infection) of 0.01 for 4 h. After two washeswith PBS, these cells were cultured at 1×10⁵ cells/ml in RPMI 1640medium supplemented with 10% FCS. The supernatant was collected on days3, 7, and 11 after viral infection. The level of the p24 antigen ofHIV-1 in each supernatant was determined with an HIV-I antigen-captureELISA test kit (DAINABOT, Tokyo, Japan) according to the manufacturer'sprotocol.

[0070] Results

[0071] Secondary Structures of tRNA^(Val) Ribozymes and their CleavageActivities in Vitro

[0072] To construct a pol III-driven ribozyme-expression cassette, wecloned a ribozyme sequence targeted to the 5′ leader sequence of HIV-1RNA (Adachi et al., 1986; Yu et al., 1993) adjacent to the sequence of atRNA^(Val) promoter, with three kinds of short linker between them(linker sequences are indicated by lowercase letters and ribozymesequences are indicated by bold capital letters in FIG. 1), to yield aset of pUCdt-Rz plasmids. The insertion of the short linkers changed theoverall structure of the transcripts and, thus, affected theaccessibility of the recognition arms of the ribozyme (recognition armsare underlined). Naturally, it is important that both the 5′ and 3′substrate-recognition arms of the ribozyme be available to the substrateso that the ribozyme can form the stem structures with the substrate RNAthat ensure subsequent cleavage of the substrate. In order to clarifythe relationship between structure and functional activity, we choselinkers that altered the extent of availability of the recognition arms.FIG. 1 shows the secondary structures of the tRNA^(Val)-ribozymes(sequences corresponding to A and B boxes are shaded), as predicted bycomputer modeling (Mulfold Biocomputing Office, Biology Department,Indiana University, Ind., USA). In one case (FIG. 1A), the linker wasinserted before the terminator sequence and restricted the flexibilityof the 3′ substrate recognition arm of the ribozyme. In addition, the 5′substrate-recognition arm was unavailable. Therefore, in the case oftRNA^(Val)-ribozyme 1 (Rz1 in FIG. 1A), both 5′ and 3′substrate-recognition arms were mostly embedded in a helical structure.tRNA^(Val)-ribozyme 2 (Rz2) has one restricted substrate-recognition armon the 5′ side. By contrast, tRNA^(Val)-ribozyme 3 (Rz3) had norestricted substrate-recognition arms and both arms were available forbinding to the substrate. Judging from the flexibility of thesubstrate-recognition arms, we might expect that the cleavage activityof Rz3 would be the highest, followed by Rz2 and Rz1 in that order. Thebase sequences of Rz1-3 are represented in SEQ ID NOS: 3, 1 and 2,respectively.

[0073] To examine whether the above ribozymes had the cleavage activitythat we predicted from their secondary structures, (FIG. 1) we firstcompared activities in vitro. Total RNA was isolated from HeLa cellsthat had been transfected with various pUCdt-Rz, plasmids that encodedthe above ribozymes (tRNA^(Val)-ribozyme). We mixed a fixed amount(based on Northern blotting data) of each ribozyme within the isolatedRNA and radiolabeled substrate RNA to initiate the cleavage reaction,and we monitored the progress of each reaction, after a 12-h incubation,on a 6% polyacrylamide/7 M urea gel (FIG. 2). As expected, the cleavageactivity of Rz3, with both recognition arms available, was the highest,followed by that of Rz2, while that of Rz1, with both recognition armsunavailable, was very low. It was, therefore, clear that the cleavageactivity of tRNA^(Val)-ribozymes in vitro could be deduced from theircomputer-generated secondary structures.

[0074] Steady-State Levels and Half-Lives of tRNA^(Val)-Ribozymes

[0075] We expected that minor structural changes would occur in theentire structure as a result of the linker sequence. Thus, the linkershould exert considerable influence on the stability of each ribozyme invivo. We compared the intracellular stability of eachtRNA^(Val)-ribozyme using two different approaches, as follows. Wecompared the steady-sate level of each transcript from HeLa cells thathad been transiently transfected with pUC-Rr (a sequence of a referencegene was added to each ribozyme-coding pUCdt-Rz plasmid to yield pUC-Rr;FIG. 3A) by Northern blotting analysis (transient expression assay). Thelevel of expression of each tRNA^(Val)-ribozyme was normalized byadjusting the amount of the transcript of the reference gene, which wasconnected, in tandem, in the same plasmid (pUC-Rr; FIG. 3A). Transcriptsof about 150 nucleotides in length (corresponding to the size ofchimeric tRNA^(Val)-ribozyme) were detected in all samples of RNA thatwe isolated from HeLa cells that had been transfected with each plasmidthat encoded a tRNA^(Val)-ribozyme. The steady-state levels of thetRNA^(Val)-ribozymes differed over a 30-fold range of concentration. Thelevel of Rz2, which was the highest, was about 26 times that of Rz1,which was the lowest, and the level of Rz3 was about 5 times that ofRz1. Since no modifications had been made in the promoter region of eachribozyme-expression cassette and, thus, since the efficiency oftranscription was assumed to be the same in each case, we postulatedthat these differences among steady-state levels of transcripts were aconsequence of the stability in vivo of each respective transcript.

[0076] As a second approach and to test the above hypothesis, weattempted to compare the stability of each transcript under morenatural, intracellular conditions. We established stable HeLatransformants that produced each tRNA^(Val)-ribozyme and measured theintracellular half-life of each transcript directly by interruptingcellular transcription with actinomycin D. As shown in FIG. 3C, the rateof degradation of Rz2 was lower than those that of Rz1 and of Rz3. Thehalf-life of Rz2 (100±10 min) was more than twice that of Rz1 (35±2min)and Rz3 (40±15 min) These results were in good agreement with theresults of the transient expression assay and supported our hypothesisthat the difference in the steady-state level of transcripts was due tothe stability in vivo of each transcript rather than to any differencesin the efficiency of transcription.

[0077] Intracellular Activities of tRNA^(Val)-Ribozymes

[0078] In order to evaluate the intracellular activities of thetRNA^(Val)-ribozymes, we performed two types of assay. We first usedeach tRNA^(Val)-ribozyme expression plasmid (pUCdt-Rz) and a target geneexpression plasmid, which encoded a chimeric HIV-1 LTR (R-U5region)-luciferase gene, to co-transfect HeLa cells. After transientexpression of both genes, in each cell lysate, we estimated theintracellular activity of each tRNA^(Val)-ribozyme by measuring theluciferase activity. The luciferase activity recorded when we used thecontrol plasmid (pUCdt), with only minimal tRNA^(Val) promoter andterminator sequences instead of the ribozyme-expression plasmid, wastaken as 100%. As shown in FIG. 4A, Rz2, which had the highest stabilityin vivo, was most effective (>60% inhibition), followed by Rz3 (>40%inhibition). Rz1 was not very effective (about 10% inhibition), asexpected from its low cleavage activity in vitro (FIG. 2B) and lowstability in vivo (FIGS. 3B and 3C).

[0079] In the second assay, only the target gene-expressing plasmid wasused to transfect stable transformants that produced almost identicallevels of tRNA^(Val)-ribozyme (stable HeLa transformants had been pickedup arbitrarily and those clones with almost identical levels ofexpression of the ribozyme were selected for these studies). In thisexperiment (FIG. 4B), with two independent stable transformants for eachribozyme, we observed a similar trend to that described in the precedingparagraph. However, in this case, the effects of all of the ribozymeswere stronger, most probably because all the transformed HeLa cellsproduced tRNA^(Val)-ribozyme constitutively. Rz2 inhibited expression ofthe target gene to a significant level, in some cases by as much as 97%.

[0080] Although Rz3 had the highest cleavage activity in vitro, itfailed to act more effectively than Rz2 in the cellular environment.These results suggest that, if a transcribed ribozyme is sufficientlystable within the cell, even if it does not have extremely high cleavageactivity, it can have a remarkable effect in vivo.

[0081] The Ability to Inhibit Replication of HIV-1

[0082] Since the above described studies demonstrated that Rz2 and Rz3might have significant cleavage activity against the sequence of HIV-1in vivo, we compared the abilities of that RNA^(Val)-ribozyme to inhibitreplication of the HIV-1. Using an HIV vector (FIG. 5; Shimada et al.,1991), we obtained stable transformants of the H9 cell line thatexpressed Rz2 or Rz3 (since Rz1 was inactive in studies described above,we above, we made no attempts to isolate stable transformants thatproduced Rz1). Cells transduced with the HIV vector without aribozyme-expression cassette (FIG. 5A) were used as a mock control. Twoindependent cell lines were used for subsequent analysis, and wedetected no obvious changes in their growth rates over a period of 11days, as compared with that of calls that did not produce eitherribozyme (data not shown). Therefore, the ribozymes were not detrimentalto host cells and probably only cleaved their target RNA with highspecificity (Kawasaki et al., 1996, 1998).

[0083] Before the virus-challenge assay, we measured the steady-statelevel of each tRNA^(Val)-ribozyme in the transduced H9 cells byquantitative RT-PCR analysis. The results of the transient expressionassay in HeLa cells shown in FIG. 3B, namely, that the difference insteady-state levels of Rz2 and Rz3 was about 5-fold, were confirmed bythe RT-PCR analysis (FIGS. 6A and 6B) Clearly, Rz2 was more stable invivo than Rz3.

[0084] When we challenged stable H9 transformants that produced atRNA^(Val)-ribozyme constitutively with HIV-1 virions, Rz2 inhibitedviral replication almost completely (about 99%), as determined on day 11post-infection (FIG. 6C). By contrast, to our surprise, Rz3 failed toinhibit viral replication at all under these experimental conditions. Inthe HIV-1 challenge assay, the difference between the effects of Rz2 andRz3 was conspicuous.

[0085] Intracellular Localization of the tRNA-Ribozymes

[0086] Since the co-localization of a ribozyme with its target isclearly an important determinant of the ribozyme's efficiency (Sullengerand Cech, 1993; Bertrand et al., 1997), it was essential to determinethe intracellular localization of tRNA^(Val)-ribozymes. Total RNA fromHeLa cells transduced with the Rz2 expression cassette was separatedinto nuclear and cytoplasmic fractions. Then, transcribed Rz2 wasdetected by Northern blotting analysis with a probe specifc for theribozyme. As shown in FIG. 7A, Rz2 was found predominantly in thecytoplasmic fraction and it was not detected to any significant extentin the nuclear fraction. The other tRNA-ribozymes (Rz1 and Rz3) werealso localized predominantly in cytoplasmic fractions (data not shown).U6 snRNA, which remains in the nucleus, was included as a control inthese studies (FIG. 7B).

[0087] Discussion

[0088] A ribozyme is a potentially useful tool for the suppression ofthe expression of specific gene since it can be engineered to act onother RNA molecules with high specificity (Uhlenbeck, 1987; Hasseloffand Gerlach, 1988). Although many trials have been successful (Ecksteinand Lilley, 1996; Turner, 1997; Scalon, 1997), it remains difficult todesign an effective ribozyme-expression system that can be used in vivo.One major challenge related to the use of ribozymes and antisense RNAsas therapeutics or genetic agents is the development of suitableexpression vectors (Jennings and Molloy, 1987; Sullenger et al., 1990;Bertrand et al., 1994, 1997; Thompson et al., 1995). Two kinds ofexpression system have been used to date, as discussed in theIntroduction, namely, the pol II system and pol III system. In thisstudy, we used the pol III system and the promoter of a human gene fortRNA^(Val) for transcription of ribozymes (Yu et al., 1993). Thispromoter is not only suitable for transcription of small RNAs, but itsuse also facilitates prediction of secondary structure by computerfolding. More importantly, it allows export of transcribed ribozymesfrom the nucleus to the cytoplasm so that the tRNA^(Val)-ribozymes canfind with their mRNA targets.

[0089] Design of Expression Cassettes

[0090] These secondary structure of a target mRNA determines itssusceptibility to ribozyme-mediated cleavage, and the ribozyme must alsofold into appropriate secondary and tertiary structures for maximalactivity. Although there is no guarantee that a computer-predictedsecondary structure really represents the corresponding structure aftertranscription, the structures predicted in this study (FIGS. 1A-1C) werewell correlated with cleavage activities in vitro (FIG. 2). In theexpression cassettes, the last seven bases of the maturetRNA^(Val)(indicated by capital letters in FIG. 1E) had been removed,without any effect on transcription, in order to block 3′-end processingof the transcript (Adeniyi-Jones et al., 1984). They were replaced by alinker (lowercase letters in FIG. 1) followed by a ribozyme (boldcapital letters). The freedom or availability of thesubstrate-recognition arms was adjusted by the linker sequence viaformation of stable stem structures in combination with the sequence ofthe tRNA^(Val) which accounted for about two-thirds of the wholesequence. Thus, it was relatively easy to predict, by computer folding,the secondary structure and the accessibility of each recognition arm.Furthermore, even if the sequence of the substrate recognition arm ischanged, as long as the same rules for predicting overall secondarystructure Dare used, it is still possible to predict, the accessibilityof recognition arms. Indeed, we have succeeded in constructing a similarribozyme-expression system for inhibition of the expression of othergenes (Kawasaki et al., 1996, 1998). Our expression system, as shown inFIGS. 1A-1C, facilitates the design of an effective ribozyme-expressioncassette.

[0091] Translocation of tRNA^(Val)-Ribozyme from the Nucleus to theCytoplasm

[0092] The ribozyme-expression cassettes shown in FIGS. 1A-1C allowedall the transcripts to be exported to the cytoplasm (FIG. 7A) where theycould find their mRNA targets, and significant inhibition by ribozymesof the expression of the target molecules was observed (FIGS. 4 and 6C).In a previous study (Bertrand et al., 1997), deletion of the last tenbases of mature tRNA^(Met) not only blocked 3′ processing but alsoinhibited the export of the transcript to cytoplasm (Tobian et al.,1985). These results suggested that 3′ processing might be linked toexport to the cytoplasm and that 3′-altered tRNA transcripts are notexported efficiently (Cotten and Birnstiel, 1989; Boelens et al., 1995).However, as demonstrated in FIG. 7, the deletion of the last seven basesof mature tRNA^(Val) did not inhibit the export of the transcripts fromthe nucleus.

[0093] A protein, designated Exportin (tRNA), which transports tRNA fromthe nucleus to the cytoplasm has recently been identified (Arts et al.,1998). Exportin (tRNA) binds RanGTP in the absence of tRNA but it doesnot bind tRNA in the absense of RanGTP. Therefore, a model for thetransport of tRNAs was proposed wherein Exportin (tRNA) associates withRanGTP first in the nucleus and then the complex binds a mature tRNAmolecule. This final complex is translocated through a nuclear porecomplex to the cytoplasm. There, the Ran-bound GTP is hydrolyzed,releasing the tRNA into the cytoplasm and allowing Exportin (tRNA) to berecycled to the nucleus (Arts et al., 1998). We do not yet know theminimal sequence or structure within a tRNA that can be recognized byExportin (tRNA). However, since the ribozymes shown in FIGS. 1A-1C weresuccessfully translocated to the cytoplasm, it is possible that theywere recognized and transported by Exportin (tRNA) despite the deletionsand alterations at the 3′ end of the natural tRNA.

[0094] It is clear, from our study, that even 32-altered tRNAtranscripts can be transported efficiently to the cytoplasm if theirsecondary structures resemble those in FIGS. 1A-1C. When we triedsimilarly to express an other kind of ribozyme (Rz4 in FIG. 1D(SEQ IDNO: 5)) in HeLa cells, the transcripts remained in the nucleus (FIG.7C). The secondary structure of Rz4 (FIG. 1D) is quite different fromthat of ribozymes Rz1, Rz2 and Rz3, which were cytoplasmic, despite thefact that not only the A and B box promoter elements (shaded boxes inFIG. 1) but also all the remaining sequence within the tRNA^(Val)segment were identical in transcripts Rz1 through Rz4. This observationsuggest that, if Exportin (tRNA) can indeed recognize the ribozymetranscript, it is unlikely that it recognizes a specific nucleotidesequence. Exportin (tRNA) might, rather, recognize some specifichigher-order structure of tRNA or some sequence within such ahigher-order structure.

[0095] Indeed, another ribozyme, constructed for other purposes, whosesecondary structure resembled that of Rz4 was found only in nuclei (datanot shown). We have constructed more than ten other ribozymes forsuppression of three other genes, keeping in mind that their secondarystructures should resemble those of Rz1 through Rz3 in FIG. 1 andadjusting linker sequences so that they might be transported to thecytoplasm. All of these ribozymes were found in the cytoplasm aftertranscription. They not only had high activities (>95% inhibition) butalso high specificity (<5% inhibition by the inactive control). Thus,cytoplasmic ribozymes based on the design shown in FIGS. 1A-1C seem veryattractive (Kawasaki et al., 1996, 1998). We should also mention thatthe secondary structures of Rossi's tRNA^(Met)-ribozymes, which remainedin nuclei and were not very active (Bertrand et al., 1997), do notresemble our active secondary structures because of their differentlinker sequence. Their structures resemble that of Rz4(computer-predicted structures not shown)

[0096] It will be of interest to determine whether ribozymes such as Rz1through Rz3 (but not Rz4 or Rossi's tRNA^(Met)-ribozymes) form complexeswith Exportin (tRNA) in the presence of RanGTP, that is, underconditions in which formation of a complex between an export receptorand its cargo would be expected (Arts et al., 1998)

[0097] Activities of tRNA^(Val)-Ribozyme in Vivo

[0098] Sullenger and Cech (1993) and Rossi's group (Bertrand et al.,1997) clearly demonstrated the importance of intracellularco-localization of ribozymes with their targets. In the case of onespecific expression cassette, both the ribozymes and its RNA target werelocated in the nucleus and the specific cleavage by the ribozyme of itstarget was detected (Bertrand et al., 1994). Thus, the criticalparameter is not the localization of the ribozyme per se but it is,rather, the ability of the ribozyme to co-localize with its target(Bertrand et al., 1997). Since various proteinaceous factors areinvolved in the intracellular processing and transport of mRNAs andsince such factors may bind promptly with mRNA immediately aftertranscription, such factors could inhibit the binding of the ribozymewith its RNA target in the nucleus. Also, in the cytoplasm, polysomesmight inhibit the binding of the ribozyme with its RNA target. Moreover,since nuclear tRNA^(Met)-ribozyme failed to inactivate a cytoplasmicmRNA that had originally been produced in the nucleus (Bertrand et al.,1997), the transport of mRNA from the nucleus to the cytoplasm seems tobe much more rapid than the attack by the nuclear tRNA^(Met)-ribozyme.One of the most critical factors determining ribozyme activity in vivoseems to be the association between the ribozyme and its target. Asignificant fraction of ribozymes must be degraded during theirtransport and also during their approach to their target site. For thisreason, co-localization of a ribozyme and its target does not, byitself, guarantee the efficacy of ribozymes in vivo.

[0099] The ribozyme Rz2, which was most stable in vivo (FIGS. 3B, 3C, 6Aand 6B), was more effective in the intracellular environment (FIG. 4)than Rz3, which had higher cleavage activity in vitro (FIG. 2). Thisdifference in activity was magnified in the HIV-1 challenge (FIG. 6C).Although cells producing the more stable Rz2 were almost completelyresistant to infection by HIV-1, other cells producing the less stableRz3 were as sensitive as control cells to infection by HIV-1. AlthoughRz2 had a half-life, that was about twice that of Rz3, it is unclear, atpresent, which structural feature(s) made Rz2 more resistant to RNases.There were six more nucleotides within the linker in Rz3, as compared toRz2, which must have influenced the higher-order structure.

[0100] The half-lives of natural tRNAs range from 50 to 60 hours (Smithand Weinberg, 1981), while that of Rz2 was only about 100 min. If thehalf-life of the tRNA-ribozyme could be prolonged, a higher inhibitoryeffect might be expected. While we still cannot predict the relativestabilities of transcripts in vivo, we can design ribozymes that can betransported into the cytoplasm by incorporating secondary structuressuch as the ones shown in FIG. 1. Since we cannot accurately predict thestability of a transcript, we usually test several constructs and, inthe case of various genes tested to date, we have always been able toobtain a cassette that can inactivate the gene of interest with >95%efficiency (Kawasaki et al., 1996, 1998)

[0101] The tRNA^(Val)-vector may be useful for expression of functionalRNAs other than ribozymes whose target molecules are localized in thecytoplasm. In our hands, tRNA^(Val)-ribozymes have consistently highactivities, at least in cultured cells. Therefore, properly designedtRNA^(Val) ribozymes appeared to be very useful as tools in molecularbiology, with potential utility in medicine as well.

[0102] Industrial Applicability

[0103] By the present invention, there is provided novel ribozymes andexpression systems therefor. The ribozyme of the present invention hashigh in vivo stability, and thereby exhibits high activity.

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1 25 1 135 RNA Artificial Sequence Description of Artificial SequenceNucleotide sequence of Rz2 1 accguugguu uccguagugu agugguuauc acguucgccuaacacgcgaa agguccccgg 60 uucgaaaccg ggcacuacaa acacaacacu gaugaggaccgaaagguccg aaacgggcac 120 gucggaaacg guuuu 135 2 141 RNA ArtificialSequence Description of Artificial Sequence Nucleotide sequence of Rz3 2accguugguu uccguagugu agugguuauc acguucgccu aacacgcgaa agguccccgg 60uucgaaaccg ggcacuacaa accaacacac aacacugaug aggaccgaaa gguccgaaac 120gggcacgucg gaaacgguuu u 141 3 128 RNA Artificial Sequence Description ofArtificial Sequence Nucleotide sequence of Rz1 3 accguugguu uccguaguguagugguuauc acguucgccu aacacgcgaa agguccccgg 60 uucgaaaccg ggcacccacacaacacugau gaguccguga ggacgaaacg ggcaccucga 120 gcgcuuuu 128 4 95 RNAArtificial Sequence Description of Artificial Sequence Nucleotidesequence of the transcript of human placental tRNA Val 4 accguugguuuccguagugu agugguuauc acguucgccu aacacgcgaa agguccccgg 60 uucgaaaccgggcggaaaca aagacagucg cuuuu 95 5 149 RNA Artificial Sequence Descriptionof Artificial Sequence Nucleotide sequence of Rz4 5 accguugguuucgguagugu agugguuauc acguucgccu aacacgcgaa aggucccccg 60 uucgaaaccgggcacccggg uggcugucac cggaagugcu uuccggucuc augaguccgu 120 gagggcgaaacagccacucg agcgcuuuu 149 6 110 DNA Artificial Sequence Description ofArtificial Sequence Sequence of a sense oligonucleotide linker 6aattcaggac tagtctttta ggtcaaaaag aagaagcttt gtaaccgttg gtttccgtag 60tgtagtggtt atcacgttcg cctaacacgc gaaaggtccc cggttcgaag 110 7 113 DNAArtificial Sequence Description of Artificial Sequence Sequence of anantisense oligonucleotide linker 7 tcgacttcga accggggacc tttcgcgtgttaggcgaacg tgataaccac tacactacgg 60 aaaccaacgg ttacaaagct tcttcttctttttgacctaa aagactagtc ctg 113 8 53 DNA Artificial Sequence Descriptionof Artificial Sequence Sequence of a sense oligonucleotide linker 8cgaaaccggg cacccgggga atataacctc gagcgctttt tttctatcgc gtc 53 9 54 DNAArtificial Sequence Description of Artificial Sequence Sequence of anantisense oligonucleotide linker 9 tcgacgcgat agaaaaaaag cgctcgaggttatattcccc gggtgcccgg tttc 54 10 23 DNA Artificial Sequence Descriptionof Artificial Sequence Sequence of an upper primer 10 cgccagggtttcccagtcac gac 23 11 101 DNA Artificial Sequence Description ofArtificial Sequence Sequence of a lower primer including the sequencesof Rz1 and a terminator 11 ctgcaggtcg acgcgataga aaaaaagcgc tcgaggtgcccgtttcgtcc tcacggactc 60 atcagtgttg tgtgggtgcc cggtttcgaa ccgggacctt t101 12 109 DNA Artificial Sequence Description of Artificial SequenceSequence of a lower primer including the sequences of Rz2 and aterminator 12 ctgcaggtcg acgcgataga aaaaaaccgt ttccgacgtg cccgtttcggtcctttcggt 60 cctcatcagt gttgtgtttg tagtgcccgg tttcgaaccg gggaccttt 10913 106 DNA Artificial Sequence Description of Artificial SequenceSequence of a lower primer including the sequences of Rz3 and aterminator 13 ctgcaggtcg acgcgataga aaaaaaccgt ttccgacgtg cccgtttcggtcctcatcag 60 tgttgtgtgt tggtttgtag tgcccggttt cgaaccgggg accttt 106 1440 DNA Artificial Sequence Description of Artificial Sequence Sequenceof a probe specific for the reference RNA 14 aaatcgctat aaaaagcgctcgaggttatg ctccccgggt 40 15 16 DNA Artificial Sequence Description ofArtificial Sequence Sequence of a probe specific for the ribozyme 15ctcatctgtg ttgtgt 16 16 20 DNA Artificial Sequence Description ofArtificial Sequence Sequence of a primer for b-actin 16 gtggccatctcttgctcgaa 20 17 18 DNA Artificial Sequence Description of ArtificialSequence Sequence of a primer for the ribozyme 17 gacctttcgg tcctcatc 1818 20 DNA Artificial Sequence Description of Artificial SequenceSequence of an upper oligonucleotide primer 18 gactacctca tgaagatcct 2019 20 DNA Artificial Sequence Description of Artificial SequenceSequence of a lower oligonucleotide primer 19 gtggccatct cttgctcgaa 2020 18 DNA Artificial Sequence Description of Artificial SequenceSequence of an upper oligonucleotide primer 20 gttatcacgt tcgcctaa 18 2118 DNA Artificial Sequence Description of Artificial Sequence Sequenceof a lower oligonucleotide primer 21 gacctttcgg tcctcatc 18 22 18 DNAArtificial Sequence Description of Artificial Sequence Sequence of aprobe specific for the ribozyme 22 acgcgaaagg tccccggt 18 23 19 DNAArtificial Sequence Description of Artificial Sequence Sequence of aprobe specific for b-actin 23 gcgggaaaat cgtgcgtga 19 24 38 RNAArtificial Sequence Description of Artificial Sequence Substrate RNAcorresponds to nucleotides 500-711 of pNL 432, namely the U5 region ofHIV-1 RNA 24 acacaacacu gaugaguccg ugaggacgaa acgggcac 38 25 17 RNAArtificial Sequence Description of Artificial Sequence Substrate RNAcorresponds to nucleotides 500-711 of pNL 432, namely the U5 region ofHIV-1 RNA 25 gugcccgucu guugugu 17

1-7. (Cancelled.)
 8. An RNA variant adopting the following secondarystructure (I), wherein said RNA variant comprises a bulge structureintroduced in the region in which hydrogen bonds form betweennucleotides 8 to 14 and nucleotides 73 to
 79.


9. The RNA variant according to claim 8 comprising a bulge structurewhich is introduced by substituting all or part of the sequence of theregion of nucleotides 73 to 79 within the nucleotide sequence of an RNAadopting secondary structure (I). 10-11. (Cancelled.)
 12. An RNAcomprising the RNA variant according claim 8 and a selected RNA chainlinked thereto.
 13. The RNA according to claim 12 wherein said selectedRNA chain is a ribozyme or an antisense RNA.
 14. The RNA according toclaim 12 wherein a bulge structure is formed with any nucleotide of anRNA chain linked to the 3′ terminus and any nucleotide of the region ofnucleotides 8 to 14 within the nucleotide sequence of an RNA adoptingsecondary structure (I).
 15. An expression vector comprising DNAencoding the RNA variant of claim 8.